Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis.

Abstract

Human chymotrypsin C (CTRC) protects against pancreatitis by degrading trypsinogen and thereby curtailing harmful intra-pancreatic trypsinogen activation. Loss-of-function mutations in CTRC increase the risk for chronic pancreatitis. Here we describe functional analysis of eight previously uncharacterized natural CTRC variants tested for potential defects in secretion, proteolytic stability, and catalytic activity. We found that all variants were secreted from transfected cells normally, and none suffered proteolytic degradation by trypsin. Five variants had normal enzymatic activity, whereas variant p.R29Q was catalytically inactive due to loss of activation by trypsin and variant p.S239C exhibited impaired activity possibly caused by disulfide mispairing. Surprisingly, variant p.G214R had increased activity on a small chromogenic peptide substrate but was markedly defective in cleaving bovine β-casein or the natural CTRC substrates human cationic trypsinogen and procarboxypeptidase A1. Mutation p.G214R is analogous to the evolutionary mutation in human mesotrypsin, which rendered this trypsin isoform resistant to proteinaceous inhibitors and conferred its ability to cleave these inhibitors. Similarly to the mesotrypsin phenotype, CTRC variant p.G214R was inhibited poorly by eglin C, ecotin, or a CTRC-specific variant of SGPI-2, and it readily cleaved the reactive-site peptide bonds in eglin C and ecotin. We conclude that CTRC variants p.R29Q, p.G214R, and p.S239C are risk factors for chronic pancreatitis. Furthermore, the mesotrypsin-like CTRC variant highlights how the same natural mutation in homologous pancreatic serine proteases can evolve a new physiological role or lead to pathology, determined by the biological context of protease function.

• Authors: András Szabó, Maren Ludwig, Eszter Hegyi, Renata Szépeová, Heiko Witt, Miklós Sahin-Tóth
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 290 Issue 28 Pg. 17282-92 (Jul 10 2015) ISSN: 1083-351X [Electronic] United States
• PMID: 26013824 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical References

• Caseins
• Recombinant Proteins
• Trypsinogen
• Chymotrypsin
• chymotrypsin C
• PRSS3 protein, human
• Trypsin

Topics

• Amino Acid Sequence
• Animals
• Caseins (metabolism)
• Cattle
• Chymotrypsin (chemistry, genetics, metabolism)
• Genetic Variation
• HEK293 Cells
• Humans
• Models, Molecular
• Molecular Sequence Data
• Mutation
• Pancreatitis, Chronic (enzymology, genetics)
• Protein Conformation
• Recombinant Proteins (chemistry, genetics, metabolism)
• Risk Factors
• Substrate Specificity
• Trypsin (chemistry, genetics, metabolism)
• Trypsinogen (chemistry, metabolism)